The red blood cell goes through six stages of development: pronormoblast, basophilic normoblast, polychromatophilic blast, orthochromic normoblast, reticulocyte, and mature red blood cell. The first four stages are normally confined to the bone marrow. The reticulocytes, however, are found in both the bone marrow and peripheral blood. Increased reticulocytes are found in hemolytic anemias, thalassemia, sideroblastic anemia, and in acute and chronic blood loss. Therefore, it is of clinical importance to analyze reticulocytes contained in a blood sample. Traditionally, reticulocyte measurement is performed manually. The process involves the smearing of a blood sample stained by new methylene blue dye on a microscope slide, followed by manual visual analysis of the individual slide. The reticulocytes are reported as percentage of the total red blood cells in the blood sample. This approach is extremely time-consuming as well as being subjective to the interpretation of the individual analyzing the slide.
In recent years, several automated methods have been developed for analysis of reticulocytes. The majority of these methods utilize fluorescent RNA stain to specifically stain reticulocytes and use fluorescence flow cytometry for detection. U.S. Pat. No. 4,985,174 to Kuroda et al. discloses a reagent containing auramine O for the fluorescent staining of reticulocytes in a sample of whole blood to permit determination of reticulocytes by means of a flow cytometer. U.S. Pat. No. 5,411,891 to Fan et al. discloses a reagent which includes an organic cationic dye for staining the reticulocytes in the blood sample and a buffer solution for maintaining pH. The reticulocytes, mean cell volume and hemoglobin concentrations are analyzed by fluorescence flow cytometer. These methods can differentiate reticulocytes from other cell populations; however, fluorescence measurement is a complex and expensive detection method.
U.S. Pat. No. 5,616,501 to Rodriquez et al. discloses a method using upper median angle light scatter (UMALS) measurement to detect stained and ghosted reticulocytes. The method precipitates intracellular RNA of the reticulocytes by incubating the blood sample with a new methylene blue dye solution, then releases hemoglobin and fixes the red blood cells by further incubating with a ghosting reagent. In the treated sample mixture, the reticulocytes have a shift in their median angle light scatter signals, which is used to differentiate the reticulocytes from mature red blood cells. This method avoids the use of costly fluorescence measurement; however, the precipitation mechanism of the staining process can be troublesome on an instrument. In addition, the critical ghosting process requires heating of the sample mixture to 41.degree. C. for an automated analysis.
On the other hand, analysis of leukocyte populations from whole blood samples is an integral and essential part of diagnostic procedures regarding a multiplicity of pathologies. The ability to analyze the major subpopulations of leukocytes in an automated manner is essential for a rapid diagnosis of a single blood sample and for the rapid processing of many samples at once.
U.S. Pat. No. 5,155,044 (to Ledis et al.) discloses a method for isolation and analysis of leukocytes from a whole blood sample, which enables differentiation of leukocytes into five subpopulations in a one-step measurement on an automated hematology analyzer. The detection technique involves a concurrent light scatter measurement and impedance measurements in both DC (direct current) and RF (radio frequency). Ledis et al. enables automated differentiation of leukocyte subpopulations, but they do not provide differentiation of reticulocytes.
U.S. Pat. No. 5,389,549 (to Hamaguchi et al.) describes a lysis reagent system and a method for differentiation of leukocytes into five subpopulations with a complex procedure. The method requires three lytic reagents, three separate sample preparations and measurements for the identity of eosinophil, neutrophil and basophil populations in addition to the lymphocyte and monocyte populations. Hamaguchi et al. enables automated differentiation of leukocyte subpopulations, but they do not provide differentiation of reticulocytes. U.S. Pat. No. 5,686,308 (to Li et al.) describes a lysing reagent system and a method for differentiation of leukocytes into five subpopulations in a one-step measurement on an automated hematology analyzer. The lytic reagent comprises a lytic reagent comprising an ethoxylated long chain amine compound and acid to adjust the pH of the lytic reagent to be within the range of 2.0 to 3.6; and a hypertonic, alkaline stabilizing reagent. This patent teaches a reagent and method for differentiation of leukocytes subpopulations, but does not teach differentiation of reticulocytes.
Based on the foregoing, there exists a need for a simple and less costly analysis method for differentiating reticulocytes.